PROJECT SUMMARY Despite significant advances in the management of metastatic HER2+ breast cancer (BC), it remains incurable. The reason for this is that cancers invariably develop resistance to standard therapies ? both cytotoxic chemotherapies and those that specifically target HER2. Modern attempts to improve upon standard therapies have largely focused on agents that inhibit HER2 downstream signaling more potently, but these have yielded only incremental benefits. Therefore, new treatment approaches are urgently needed. Recently it has become clear that HER2+ BCs are immunogenic. HER2 is a strong tumor antigen, and a proportion of HER2+ BCs harbor a lymphocytic infiltrate, which predicts for improved outcomes. In addition, anti-HER2 antibodies exert their effects in part by stimulating immune effector cells. Collectively, these facts provide rationale for testing immunotherapy in HER2+ metastatic BC. Our co-investigator Dr. Loi recently conducted a phase II study of trastuzumab and pembrolizumab (a PD-1 inhibitor) in patients with HER2+ metastatic BC (PANACEA). The study met its primary endpoint and thus provides proof-of-principle for the use of immunotherapy in HER2+ disease ? however, only a small minority of patients benefited. In Project 2, we will therefore study two novel therapeutic approaches designed to boost the anti-tumor immune response against HER2+ BC further: the use of CDK4/6 inhibitors, given together with trastuzumab and PD-1 blockade (Aim One); and the addition of PD- L1 blockade and a 4-1BB agonist to chemotherapy and trastuzumab (Aim Two). Both regimens are rationally designed, based on our convincing preclinical data showing that these approaches markedly amplify anti-tumor immunity. In each Aim, we will perform a randomized, multicenter phase II clinical trial to determine the efficacy of these novel approaches. Each Aim will also employ a ?co-clinical trial? model, with mouse studies running in parallel to human trials. The animal experiments will be performed using our state-of-the-art transgenic model of human HER2- driven mammary carcinoma (MMTV-rtTA/tetO-HER2). Our mouse studies incorporate cutting- edge technologies to understand the mechanisms of activity for these novel immunotherapy approaches, as well as detailed studies of resistance mechanisms (including next-generation sequencing and multiplexed immunofluorescent profiling of tumor tissue). Meanwhile, the trials involve serial collection of tumor biopsies and blood samples. Biospecimens from mice and patients will be analyzed in parallel, with each informing the other. Ultimately, these studies will: (1) characterize the immune landscape of advanced HER2-positive BC in unprecedented detail; (2) determine whether either of the two novel approaches is an effective clinical strategy; (3) establish cellular mechanisms of activity for each regimen; and (4) explore mechanisms of therapeutic resistance. This work has the potential to uncover new therapies that enhance immune responses against HER2-positive BC, and thus significantly improve patient outcomes.